S. I. Belikov

Identification and first insights into the structure and biosynthesis of chitin from the freshwater sponge Spongilla lacustris

This work demonstrates that chitin is an important structural component within the skeletal fibers of the freshwater sponge Spongilla lacustris. Using a variety of analytical techniques ((13)C solid state NMR, FT-IR, Raman, NEXAFS, ESI-MS, Morgan-Elson assay and Calcofluor White Staining); we show that this sponge chitin is much closer to α-chitin, known to be present in other animals, than to β-chitin. Genetic analysis confirmed the presence of chitin synthases, which are described for the first time in a sponge. The presence of chitin in both marine (demosponges and hexactinellids) and freshwater sponges indicates that this important structural biopolymer was already present in their common ancestor.

First report on chitinous holdfast in sponges (Porifera).

A holdfast is a root- or basal plate-like structure of principal importance that anchors aquatic sessile organisms, including sponges, to hard substrates. There is to date little information about the nature and origin of sponges’ holdfasts in both marine and freshwater environments. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within holdfasts of the endemic freshwater demosponge Lubomirskia baicalensis. Using a variety of techniques (near-edge X-ray absorption fine structure, Raman, electrospray ionization mas spectrometry, Morgan–Elson assay and Calcofluor White staining), we show that chitin from the sponge holdfast is much closer to α-chitin than to β-chitin. Most of the three-dimensional fibrous skeleton of this sponge consists of spicule-containing proteinaceous spongin. Intriguingly, the chitinous holdfast is not spongin-based, and is ontogenetically the oldest part of the sponge body. Sequencing revealed the presence of four previously undescribed genes encoding chitin synthases in the L. baicalensis sponge. This discovery of chitin within freshwater sponge holdfasts highlights the novel and specific functions of this biopolymer within these ancient sessile invertebrates.

The relationship between the structure of the tick-borne encephalitis virus strains and their pathogenic properties.

Tick-borne encephalitis virus (TBEV) is transmitted to vertebrates by taiga or forest ticks through bites, inducing disease of variable severity. The reasons underlying these differences in the severity of the disease are unknown. In order to identify genetic factors affecting the pathogenicity of virus strains, we have sequenced and compared the complete genomes of 34 Far-Eastern subtype (FE) TBEV strains isolated from patients with different disease severity (Primorye, the Russian Far East). We analyzed the complete genomes of 11 human pathogenic strains isolated from the brains of dead patients with the encephalitic form of the disease (Efd), 4 strains from the blood of patients with the febrile form of TBE (Ffd), and 19 strains from patients with the subclinical form of TBE (Sfd). On the phylogenetic tree, pathogenic Efd strains formed two clusters containing the prototype strains, Senzhang and Sofjin, respectively. Sfd strains formed a third separate cluster, including the Oshima strain. The strains that caused the febrile form of the disease did not form a separate cluster. In the viral proteins, we found 198 positions with at least one amino acid residue substitution, of which only 17 amino acid residue substitutions were correlated with the variable pathogenicity of these strains in humans and they authentically differed between the groups. We considered the role of each amino acid substitution and assumed that the deletion of 111 amino acids in the capsid protein in combination with the amino acid substitutions R16K and S45F in the NS3 protease may affect the budding process of viral particles. These changes may be the major reason for the diminished pathogenicity of TBEV strains. We recommend Sfd strains for testing as attenuation vaccine candidates.

Monophyletic origin of freshwater sponges in ancient lakes based on partial structures of COXI gene

The phylogenetic relationships between freshwater sponges (families Lubomirskiidae, Spongillidae and Potamolepidae) are still poorly understood. Partial sequences of the cytochrome oxidase subunit I (COXI) gene were obtained from seven freshwater sponge species and one marine sponge. Phylogenetic analyses showed that all the freshwater sponge species formed one common clade and they appeared to be closely related. These results indicate that freshwater sponges are of monophyletic origin. Potamolepidae and Spongillidae are situated at the base of the tree and are assumed to be phylogenetically older than Lubomirskiidae.

Comparative study of two protein-coding regions of mitochondrial DNA from three endemic sculpins (cottoidei) of Lake Baikal

SummaryTwo protein-coding regions (cytochrome b, ATPase 8, and part of ATPase 6) from mitochondrial DNA of Cottus kessleri, Cottocomephorus grewingki, and Cottocomephorus inermis-Baikalian endemic sculpins—were amplified via polymerase chain reaction, and sequenced. Two novel primers-L8352 (5′-TAAAGATTGGTGAC TCCCAACCACC) and H8773 (5′-GTAGGGAGT AAGCCCAATATGTT)-were used for the latter region. Phylogenies suggested by sequence divergence of the genes of ATPases appeared to be different from those computed from data for cytochrome b. The time of species branching was estimated as 1–2 million years (Myr) on the basis of merged sequences. Hence, members of the Baikalian cottoid species flock are much more distant from each other than members of the cichlid fish flocks of the great lakes of Africa (0.2 Myr). Topology of the phylogenetic tree does not contradict the relationships derived from morphological data. However, genetic distances suggest that C. grewingki and C. inermis are not sister species, contrary to general belief.

Comprehensive assessment of the genetics and virulence of tick-borne encephalitis virus strains isolated from patients with inapparent and clinical forms of the infection in the Russian Far East.

We analyzed the genetics and virulence of 35 strains of TBEV isolated from patients with different forms of the infection living in the southern Far East region of Russia. The results of moleculargenetics studies of the TBEV strains showed that most of the strains that cause inapparent infections form a single cluster (I) with the Oshima 5-10 strain from Japan on the phylogenetic tree. A comparison of the amino acid sequences of the viral polyproteins of the studied strains identified 17 amino acid residues distributed unevenly across the polyprotein that distinctly differed between the clusters of inapparent and virulent strains. We detected additional substitutions in the NS1 and NS5 proteins. These substitutions might influence the pathogenic potential of the strains. Using a model of inbred mice of different ages, we examined the virulence of these strains and showed the different pathogenic potentials of strains belonging to different clusters.

Molecular identification and phylogeny of Dermacentor nuttalli (Acari: Ixodidae)

Dermacentor nuttalli is an epidemiologically important tick in Palearctic Asia which transmits several infectious diseases including tularemia, North Asian tick-borne rickettsiosis, Lyme disease and tick-borne encephalitis. The genetic specificity and phylogeny of D. nuttalli from four geographic localities in Eastern Siberia were characterized using the mitochondrial (mt) 16S ribosomal RNA (rRNA) gene and internal transcribed spacer 2 (ITS2). Low genetic diversity was observed in the populations of ticks distributed from South Siberia to North China. From 11 detected mt 16S haplotypes, one was found in all populations, whereas the others were restricted to specific localities. These results suggested that the genetic structure of D. nuttalli represents integrated populations with no geographic isolation across the distribution area. The phylogenetic reconstructions inferred from the mt 16S rRNA gene and ITS2 were in agreement and showed a distinct D. nuttalli clade within a monophyletic Eurasian lineage of Dermacentor sp.

Formation of Spicules During the Long-term Cultivation of Primmorphs from the Freshwater Baikal Sponge Lubomirskia baikalensis

Sponges (phylum Porifera) are phylogenetically ancient Metazoa that use silicon to form their skeletons. The process of biomineralization in sponges is one of the important problems being examined in the field of research focused on sponge biology. Primmorph cell culture is a convenient model for studying spiculogenesis. The aim of the present work was to produce a long-term primmorph culture from the freshwater Baikal sponge Lubomirskia baikalensis (class Demospongiae, order Haplosclerida and family Lubomirskiidae) in both natural Baikal water and artificial Baikal water to study the influence of silicate concentration on formation and growth of spicules in primmorphs. Silicate concentration plays an important role in formation and growth of spicules, as well as overabundance of silica leads to destruction of cell culture primmorphs. We also found that the composition of chemical elements (Si, O, C, and Na) varied along the length of growing spicules at cultivation in different media. The long-term culture of Baikal sponge primmorphs will be necessary for further investigations, and this system may serve as a powerful in vitro model to study spiculogenesis in Baikal siliceous sponges during the early stages of intracellular spicule formation to identify genes that affect biomineralization.

[Coding nucleotide sequences of tick-borne encephalitis virus strains isolated from human blood without clinical symptoms of infection].

Genomes of four tick-borne encephalitis virus strains, isolated from the blood of the individuals after tick bites and causing no clinical symptoms of infection, were characterized. Analysis of translated polypeptides revealed 21 amino acid positions typical of this group of strains and distinguishing them from the other tick-borne encephalitis virus strains of Far Eastern subtype examined earlier. Only three mutations led to substantial amino acid changes, which probably could affect the infection process severity. It is suggested that two associated mutations, deletion of amino acid 111 in the capsid protein C and substitution (Ser1534 → Phe) in the NS3 protein influence strictly coordinated polyprotein processing, disturbing correct arrangement of viral particles. This process can result in the development of defect viral particles, containing no RNA. Mutation (Ser917 → Gly) in nonstructural protein NS1 results in the substitution of hydrophilic amino acid, specific to highly virulent strains, by the hydrophobic one. This could influence the effectiveness of viral replication complex, thereby affecting the infectivity of tick-borne encephalitis virus strains.

Identification of silicateins in freshwater sponge Lubomirskia baicalensis

Siliceous spicules of Baikal freshwater sponge Lubomirskia baicalensis contain several proteins, including silicateins. Analysis of a L. baicalensis cDNA library revealed four different mRNAs coding for proteins related to marine sponge silicatein α (α1, α2, α3, and α4). The intron-exon structure was determined forthe genomic α1 silicatein gene. The gene is 1988 bp from the initiation to the termination codon and consists of six intron (total size 1007 bp) and seven exons (total size 981 bp). Mass spectrometry of a tryptic digest of spicule proteins revealed peptides of two silicateins α.